Efficacy of Advanced Odomos repellent cream ( N , N-diethyl-benzamide ) against mosquito vectors

Methods: Bioassays were conducted to assess the repellency of Advanced Odomos and DEET creams against Anopheles stephensi and Aedes aegypti. Their efficacy was tested on human volunteers applied with different concentrations of test creams ranging from 1 to 12 mg/cm2 and by exposing them to mosquitoes at hourly intervals. Field evaluation was also carried out to test the duration of protection of the test creams against Anopheles and Aedes mosquitoes during whole night and day time collections, respectively on human volunteers. Mosquito collections were done using torch light and aspirator.

Background & objectives: Repellents are commonly used personal protection measures to avoid mosquito bites.In the present study, Advanced Odomos cream (12% N, N-diethyl-benzamide) was tested for its efficacy against mosquitoes in comparison to DEET (N,N-diethyl-3-methyl benzamide).
Methods: Bioassays were conducted to assess the repellency of Advanced Odomos and DEET creams against Anopheles stephensi and Aedes aegypti.Their efficacy was tested on human volunteers applied with different concentrations of test creams ranging from 1 to 12 mg/cm 2 and by exposing them to mosquitoes at hourly intervals.Field evaluation was also carried out to test the duration of protection of the test creams against Anopheles and Aedes mosquitoes during whole night and day time collections, respectively on human volunteers.Mosquito collections were done using torch light and aspirator.
Results: Complete (100%) protection was achieved at 10 mg/cm 2 cream formulation of Advanced Odomos (1.2 mg a.i/cm 2 ) dose against An.stephensi and 12 mg/cm 2 (1.44 mg a.i./cm 2 ) against Ae.aegypti on human baits.There was no statistically significant differences in per cent protection against mosquito bites between Advanced Odomos and DEET cream (P>0.05) in respective doses.Complete protection up to 11 h was observed against Anopheles mosquitoes during whole night collections and up to 6 h against Ae.aegypti in day time collections.No adverse reactions such as itching, irritation, vomiting, nausea, etc. were reported by the volunteers.
Interpretation & conclusions: Advanced odomos cream applied at 10 mg/cm 2 concentration provided 100% protection from Anopheles mosquitoes up to 11 h whereas about 6 h protection was recorded against Ae.aegypti.The laboratory and field trials indicate that for longer protection against Anopheles mosquitoes 10 mg/cm 2 will be appropriate and in case of Ae. aegypti more than 10 mg/cm 2 application is required for complete protection.In conclusion, the Advanced Odomos cream was comparable to the known repellent cream DEET for prolonged protection against malaria and dengue vectors.

Key words
Advanced Odomos -Anopheles culicifacies -An.stephensi -Aedes aegypti -DEET -repellency mosquito-borne diseases.Personal protection is one of the established methods to prevent mosquito bites.Use of household mosquito repellents by individuals and communities also play an important role particularly against day-time biting mosquito species such as Aedes, the vector of dengue, dengue haemorrhagic fever and chikungunya and during dusk time against other mosquitoes including malaria vectors 1 .Repellents that are currently available are either synthetic chemicals, such as DEET (N,N-diethyl-3-methyl benzamide, previously called N,N-diethyl-m-toluamide), or plant derived chemicals such as Citronella.DEET is the most effective and best studied insect repellent currently in the market [1][2][3][4][5][6] .Although DEET is very good repellent, the non-availability of 3-methylbenzoic acid from indigenous sources has made it expensive chemical in India 7 .The alternative, dimethylphthalate (DMP), also need to be replaced in India because of its low efficacy 7 .In view of this, significant efforts have been made toward developing longer-lasting and/or more cosmetically accepted repellent products.In the present study, Advanced Odomos cream developed by M/s.Balsara Home Products Ltd. (now M/s.Dabur Research Foundation, Sahibabad, UP) was evaluated for its efficacy under laboratory and field conditions against Anopheles and Aedes mosquitoes.DEET was taken as gold standard for comparison.
The study was undertaken during December 2005 to November 2006 in the laboratory and in field conditions of the respective habitats of Anopheles spp and Aedes aegypti as per guidelines prescribed in 'Protocols for uniform evaluation of insecticides for use in vector control' 8 with some modifications.The institutional Human Ethical Committee approved the study.

Laboratory tests on human baits:
The tests were conducted at the National Institute of Malaria Research, New Delhi, in a laboratory maintained at 27 ± 2 o C and 60-70 per cent relative humidity.The light intensity was regulated at 300-500 lux for testing against laboratory colonized Ae. aegypti, a day biting mosquito and at about 20-50 lux for An.stephensi, a night biter.Laboratory reared 3-5 day old female mosquitoes starved for overnight were used for bioassays.Testing was done in the laboratory maintained at standard temperature and humidity against laboratory colonized strains of An. stephensi and Ae.aegypti in cage bioassays using human volunteers.Repellent cream was applied on an approximate 100 cm 2 area of one of the fore arm (wrist to elbow) of human volunteer.Remaining exposed area was covered with a cloth sleeve.Different concentrations -4, 8, 10, and 12 mg/cm 2 of whole cream base (0.48, 0.96, 1.2 and 1.44 mg/cm 2 active ingredient) were tested.Tests were continued on each species until 100 per cent protection was obtained in four hours of exposure.Exposures of hand and arm applied with different doses of the repellent cream and control were made separately in different cloth cages containing 100 three-day old mosquitoes of each species that were pre-starved for overnight.Inserting an untreated hand into the cage before the start of each exposure ensured the propensity of mosquito for biting.Hands of the volunteer treated with different concentrations of Advanced odomos cream, DEET cream and a base cream without active ingredient (control) were introduced in different cages.Five minute landing counts were made manually at 0, 1, 2, 3 and 4 hours.Volunteers were asked to remove treated hands between the exposure intervals.Number of mosquitoes landing on different doses of repellent cream and control at different intervals was scored manually.The experiment was replicated four times for each concentration of the test cream and controls.Per cent protection for each concentration and species was calculated using the formula mentioned below.The time between the initial introduction of repellent treated hand and the first confirmed landing followed by the second successive landing was recorded as the protection time and the test was terminated after the second landing of the mosquito.
Four night /day collections were made during July to September 2006.In the village/locality, six houses were selected, two each for Advanced Odomos, INDIAN J MED RES, APRIL 2011 DEET and control (cream without active ingredient).The houses and volunteers were selected randomly and volunteers (baits) as well as insect collectors were blinded for the doses and repellent creams.Preinformed and free consent was obtained before hand from the volunteers participating as baits.The cream was applied on exposed parts as per the specified dose (face and legs below the knee to ankle) and the remaining parts were covered with thick clothes.The volunteers were allowed to relax on cot throughout the night.One insect collector covered completely with clothes, gloves and head mask was deployed to collect mosquitoes landing on the volunteer.The mosquitoes landing on the bait were collected before engorging.In addition, mosquitoes that entered the room having bait and resting inside were also collected.The insect collector was rotated after every two hours.
Mosquitoes collected on baits were identified into species using standard keys in the laboratory [9][10] .The time between application of repellent and the second successive landing on the bait was recorded as the protection time and the average of eight replicates was calculated as the mean protection time.The per cent protection data were transformed using arcsine and subjected to statistical analysis to find out the differences between means at 0.05 per cent level of significance between Advanced Odomos and DEET creams.The data were compared using Student t-test for differences between means at 0.05 per cent level of significance.

Results
Laboratory evaluation of Advanced Odomos and DEET creams against An.stephensi and Ae.aegypti on human baits is shown in Table I.On human baits, 4, 8, and 10 mg/cm 2 doses were tested against An.stephensi and in case of Ae. aegypti 12 mg/cm 2 dose was also tested additionally.100 per cent protection was achieved at 10 mg/cm 2 dose against An.stephensi and 12 mg/ cm 2 against Ae.aegypti up to 4 h of observation.There was no statistically significant differences in per cent protection against mosquito bites between Advanced Odomos and DEET creams in respective doses.
Results of field trials of Advanced Odomos and DEET creams during whole night bait collections against different species of mosquitoes are shown in Table II.Per cent protection with Advanced odomos cream applied at 10 mg/cm 2 was 100 per cent against An.culicifacies, An. stephensi, An. anuularis and An.subpictus; and 98.8 per cent against Cx.quinquefasciatus up to 11 h of observation time.The per cent protection with DEET cream applied at the same dosages was 100 per cent against all the above mentioned species.
The results of field trials against Ae.aegypti showed 92.5 per cent protection (repellency effect) with Advanced Odomos cream and 96.2 per cent protection with DEET cream applied at 10 mg/cm 2 .Results further revealed that Odomos cream provided protection up to 6.2 + 0.4 h and DEET cream provided protection up to 6.75 ± 0.2 h.All the volunteers were provided with a questionnaire regarding their opinion on protection from mosquito bites and side-effects, if any, such as itching or other aberrations of the skin and they did not report any side effects of application of both creams.

Discussion
A number of laboratories are screening different compounds of different nature and origin for mosquito repellent properties with the aim of developing a formulation giving similar or better protection than DEET, which is used since 1950s.Although effective, DEET is not the ideal product, as allergic and side effects have been reported 11 and its solvents can damage plastics and other synthetic materials.
Osimitz and Grothaus 12 concluded that 'DEET can be used with the confidence that the risk of serious adverse effects is very low'.The controlled -release insect repellent formulations for topical use that will provide extended protection against biting arthropods, be safe and pleasant to use and be compatible with plastics, synthetic fabrics and similar materials are most important for prolonged protection against bites of arthropod insects.
In laboratory tests, both DEET and Advanced Odomos creams provided 100 per cent protection when applied at 10 mg/cm 2 dose against An.stephensi and at 12 mg/ cm 2 against Ae.aegypti.In field tests on human volunteers, both creams provided complete protection up to 11 h against Anopheles mosquitoes and about 6 h against Ae.aegypti.The present study results are in conformity with earlier reports.In one laboratory study carried out elsewhere 13 , 50 per cent DEET provided up to 4 h protection against Ae.aegypti mosquitoes and increasing the concentration to 100 per cent provided only one hour additional protection.In another study 14 , 12.5 per cent DEET provided over 6 h of protection against Ae.albopictus; doubling DEET concentration to 25 per cent increased the protection time only to about 8 h.Frances et al 15 reported contrasting results between laboratory and filed efficacy of repellent formulations against Anopheles mosquitoes.Another study 16 reported that three brands of DEET formulations provided >99 per cent protection from bites of Ae. taeniorhyncus in field conditions and >95 per cent protection against An.quadrimaculatus in cage studies.Kalyanasundaram & Mathew 2 reported about 6-6.5 h of protection with diethylbenzamide against Aedes aegypti.In the present study also a protection time of about 6 h was observed with Advanced Odomos cream against Ae.aegypti.In conclusion, the present study results demonstrated that the efficacy of Advanced Odomos and DEET creams was comparable in providing protection against mosquito bites for longer duration.

Table I .
Per cent protection against mosquito species exposed to human volunteers applied with Advanced Odomos and DEET creams in cage bioassays in laboratory conditionsValues are mean ± SD of four replicate tests.Figures in parentheses are duration of protection in hours (the time at which second confirmatory bite was recorded), ND, not done

Table II .
Field evaluation of Advanced Odomos and DEET creams (10 mg/cm 2 ) against mosquitoes on human baits Data of eight replicates.*Day time collections were made in case of Ae. aegypti from 0700 to 1800 h